First Report of Bean Leafroll Virus in Pea and Chickpea, in Canada.

Bean leafroll virus (BLRV; Bean leafroll virus), a single-stranded RNA virus in the genus Luteovirus, is phloem-limited and primarily transmitted by aphids in a non-propagative, persistent manner (Rashed et al., 2018; Kidanemariam and Abraham, 2023). BLRV infects various legumes and has been reported from major pulse-growing regions worldwide (Agindotan et al., 2019) but not in the Canadian Prairies. Its impact on crop yield varies with plant and virus genotypes and the timing of infection. Some pea fields have experienced disease rates of up to 80% (Clement et al., 2020; Hampton, 1983). Throughout the 2022 growing season (June and July), pulse fields from across Saskatchewan were randomly selected and surveyed, and symptomatic plants demonstrating leaf yellowing and chlorosis were collected and stored at -80°C before processing. Observed symptoms included necrotic spots, chlorosis, leaf mottling, leaf rolling in peas, severe bright yellowing, and leaf marginal necrosis in chickpeas. BLRV detection was performed on 35 leaves of the collected samples using both Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse transcription polymerase chain reaction (RT-PCR). ELISA testing followed the manufacturer's protocol using a commercial kit (Nano Diagnostics, San Jose, CA, USA). Total RNAs were extracted from the frozen samples using TRIzol (Invitrogen, Carlsbad, CA, USA). For the detection of the diverse BLRV isolates, sequences of various isolates were aligned and primers were specifically designed in-house, targeting the virus's highly conserved regions on the GP3 and 3' UTR (see Supplementary material). Additional primers were also designed targeting coat protein (CP) coding regions which were previously used for BLRV detection (Agindotan et al. 2019; Larsen & Webster 1999). PCR testing of 35 symptomatic samples including 12 pea plants and 23 chickpea plants, identified the presence of BLRV in two symptomatic samples, one each from a field pea (Pisum sativum L. var. CDC Inca) and a desi-type chickpea (Cicer arietinum L. var. CDC Leader). The infected pea and chickpea samples were found in Saskatoon, SK (Coordinates: 52°9'27''N,106°34'14"W), and the Leader area, southwest of Saskatchewan, SK (Coordinates: 50°52'14"N,109°23'11"W), respectively. PCR amplicons were purified and sent for Sanger sequencing. The reads were assembled to generate 1666 and 323 nucleotides from pea and chickpea, respectively, with a minimum of 2X coverage. Partial nucleotide sequences of the BLRV isolates obtained from pea (PsSK1) and chickpea (CaSK1) (GenBank accession numbers: PP240429, PP266588) showed (1521/1574 bp) 96.63% and (316/323 bp) 97.83% similarity with a BLRV reference isolate sequence (NC_003369) and to an isolate from Argentina (KR261610) which was reported on Medicago sativa L. with (1555/1574 bp) 98.79% and (319/323 bp) 98.76% similarity, correspondingly. Both infected samples were confirmed to be BLRV-infected through the ELISA and exhibited a high interaction ratio (PsSK1: 0.319 and CsSK1: 0.245) compared to a positive control (0.292) after 30 minutes as measured at 450 nm. This is the first report of BLRV in the pulse-growing region of the Canadian Prairies. In Saskatchewan, there is no history of BLRV despite the large amount of area growing susceptible crops. Therefore, the survey project that this study was part of was not intended to evaluate the severity of BLRV but rather to determine if there is any virus present that might have been overlooked. The samples were therefore taken randomly, with a focus on the number of fields and geographic coverage rather than focusing on multiple plants per field. Moreover, fields were not chosen based on symptoms but rather at random. Although, plants within fields were chosen because they displayed symptoms. Typically, a disease note includes estimates of severity and potential risk; however, that is not possible for this study. Rather, the fact that it was detected indicates a greater risk than previously perceived, since it was assumed that BLRV was not present. These findings highlight the need for further research on the virus's current status, its impact on crop production, and the resistance of pulse varieties grown in Saskatchewan.